Metabolic inhibitors against brassinosteroids

ABSTRACT

A brassinosteroid metabolism inhibitor, which comprises as an active ingredient a compound represented by the following formula (I) or (II):                  
 
wherein R 1  and R 2  independently represent hydrogen atom or a lower alkyl group, R 3  represents a hydrogen atom, a lower alkyl group, or a lower alkoxyalkyl group, R 4  represents a phenyl group which may be substituted, X represents a single bond or —CH 2 —, R 11  represents a lower alkyl group, a lower alkenyl group or a phenyl group which may be substituted, R 12  represents a lower alkyl group or a phenyl group which may be substituted, and R 13  represents a phenyl group which may be substituted, or a salt thereof.

TECHNICAL FIELD

The present invention relates to an inhibitor against brassinosteroidmetabolism.

BACKGROUND ART

Brassinosteroids have been recently recognized as a new class of planthormones through the combination of molecular genetics and researches onbiosyntheses (Yokota, Trends in Plant Sci., 2, pp. 137–143, 1997). Sincethe chemistry of brassinosteroids was established, biological activitiesof these homologues have been extensively studied, and their notableactions on plant growth have been revealed, which include elongation ofstalks, growth of pollen tubes, inclination of leaves, opening ofleaves, suppression of roots, activation of proton pump (Mandava andAnnu. Rev. Plant Physiol. Plant Mol. Biol., 39, pp. 23–52, 1988),acceleration of ethylene production (Schlagnhaufer et al., Physiol.Plant, 61, pp. 555–558, 1984), differentiation of vessel elements(Iwasaki et al., Plant Cell Physiol., 32, pp. 1007–1014, 1991; Yamamotoet al., Plant Cell Physiol., 38, pp. 980–983, 1997), and cell extension(Azpiroz et al., Plant Cell, 10, pp. 219–230, 1998).

Furthermore, mechanisms and regulations of physiological actions ofbrassinosteroids have been being revealed by variety of studies on theirbiosynthesis (Clouse, Plant J. 10, pp. 1–8, 1996; Fujioka et al.,Physiol. Plant, 100, pp. 710–715, 1997). At present, 40 or morebrassinosteroids have been identified. Most of C28-brassinosteroids arecommon vegetable sterols, and they are considered to be biosynthesizedfrom campesterol, which has the same carbon side chain as that ofbrassinolide.

Several Arabidopsis mutants which show characteristic dwarfism have beenisolated, i.e., dwfl: Feldman et al., Science, 243, pp. 1351–1354, 1989;dim: Takahashi et al., Genes Dev., 9, pp. 97–107, 1995; and cbb1:Kauschmann et al., Plant J., 9, pp. 701–703, 1996. Their structuralphotomorphogenesis and dwarfism (cpd: Szekeres et al., Cell, 85, pp.171–182, 1997) and de-etiolation (det2: Li et al., Science, 272, pp.398–401, 1996; Fujioka et al., Plant Cell, 9, pp. 1951–1962, 1997) areknown. The mutants have deficiencies in the brassinosteroid biosyntheticpathway. Furthermore, a dwarf mutant of Pisum sativum was recentlycharacterized, and the mutant was reported to be a brassinosteroiddeficient mutant (Nomura et al., Plant Physiol., 113, pp. 31–37, 1997).In these plants, use of brassinolide is known to negate severe dwarfismof the mutants. Although these findings suggest that roles ofbrassinosteroids are indispensable for growth and development of plants,an effective tool other than the analysis of mutants has been desired toelucidate physiological importance of brassinolide.

As seen in researches of gibberellin action, specific inhibitors againstthe biosynthesis are generally very effective tools for elucidatingphysiological functions of endogenous substances. Specific inhibitorsagainst brassinosteroid biosynthesis are expected to provide a new toolfor understanding the functions of brassinosteroids. Uniconazole is apotent plant growth regulator (PGR) which inhibits oxidation employed bycytochrome P-450 in the steps of the gibberellin biosynthesis froment-kaurene to ent-kaurenoic acid. Yokota et al. observed slightreduction of the amount of endogenous castasterone as a side effect ofthat compound (Yokota et al., “Gibberellin”, Springer Verlag, New York,pp. 339–349, 1991). Although uniconazole in fact inhibitsdifferentiation of vessel elements induced by brassinolide (Iwasaki etal., Plant Cell Physiol., 32, pp. 1007–1014, 1991), its inhibitoryaction against brassinolide is considered to be no more than anincidental action, because uniconazole essentially inhibits thegibberellin biosynthesis.

Several mutants deficient in biosynthetic enzymes are known forArabidopsis, and their morphologic changes are unique to mutants withdeficiency in the brassinosteroid biosynthesis. Therefore, the inventorsof the present invention conducted intensive search for a compoundinducing the morphologic changes unique to the mutants with thebrassinosteroid biosynthesis deficiency to find a specific inhibitoragainst the brassinosteroid biosynthesis. As a result, they found thattriazole compounds such as4-(4-chlorophenyl)-2-phenyl-3-(1,2,4-triazoyl)butan-2-ol had the desiredinhibitory action (Japanese Patent Unexamined Publication (Kokai) No.2000-53657).

Meanwhile, it has been reported that genetic regulation of thebrassinosteroid metabolism makes plants highly sensitive tobrassinosteroids, and thus an effect of brassinosteroid administrationis markedly enhanced (Neff, M. M., et al., Proc. Natl. Acad. Sci., USA,96, pp. 15316–23, 1999). It may be possible to regulate plant growth byusing this method. However, this technique has a problem that itsapplication to an arbitrary plant at an arbitrary time is difficult.Further, it is known that plant growth can be regulated by administeringbrassinosteroids themselves to plants, and hence their yield and stressresistance can be enhanced. However, since brassinosteroids areexpensive, their application as agricultural chemicals is difficult. Itis expected that, if the brassinosteroid metabolism can be inhibited bya chemical agent, sensitivity of plants to brassinosteroids can beeasily enhanced. However, no substance which inhibits thebrassinosteroid metabolism has hitherto been known, and thus this methodcannot be utilized.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide an inhibitor againstthe brassinosteroid metabolism. The inventors of the present inventionconducted various studies to achieve the aforementioned object. As aresult, they found that triazole compounds such as difenoconazole actedas a brassinosteroid metabolism inhibitor. The inventors furthercontinued their researches, and found that the compounds represented bythe following formula (I) or (II) inhibited the brassinosteroidmetabolism, and these compounds had a regulatory action on plant growthby inhibiting the brassinosteroid metabolism. Thus, the presentinvention was achieved.

The present invention thus provides a brassinosteroid metabolisminhibitor which comprises, as an active ingredient, a compoundrepresented by the following formula (I) or (II) or a salt thereof:

wherein R¹ and R² independently represent hydrogen atom or a lower alkylgroup, R³ represents hydrogen atom, a lower alkyl group, or a loweralkoxyalkyl group, R⁴ represents a phenyl group which may besubstituted, X represents a single bond or —CH₂—, R¹¹ represents a loweralkyl group, a lower alkenyl group, or a phenyl group which may besubstituted, R¹² represents a lower alkyl group or a phenyl group whichmay be substituted, and R¹³ represents a phenyl group which may besubstituted.

As preferred embodiments of the present invention, there are providedthe aforementioned metabolism inhibitor, wherein R¹ and R² are hydrogenatoms, R³ is methyl group, R⁴ is 4-(4-chlorophenyl)oxy-2-chlorophenylgroup or biphenyl-4-yl group, and X is a single bond; the aforementionedmetabolism inhibitor, wherein R¹ and R² are hydrogen atoms, R³ isn-propyl group or methoxymethyl group, R⁴ is chlorophenyl group ormethoxyphenyl group, and X is a single bond; and the aforementionedmetabolism inhibitor, wherein R¹¹ is methyl group, R¹² is phenyl group,and R¹³ is 4-chlorophenyl group. As the brassinosteroid, brassinolide ispreferred.

As another aspect of the present invention, there are provided a methodfor inhibiting brassinosteroid metabolism in a plant, which comprisesthe step of applying the compound represented by the aforementionedformula (I) or (II) or a salt thereof to a plant; and a method forregulating plant growth by inhibiting the brassinosteroid metabolism byusing a metabolism inhibitor which comprises the compound represented bythe aforementioned formula (I) or (II) or a salt thereof as an activeingredient.

According to a further aspect of the present invention, there isprovided a plant growth regulator which comprises the aforementionedbrassinosteroid metabolism inhibitor. This plant growth regulator has aninhibitory action against the brassinosteroid metabolism and can be usedas a plant growth regulator for, for example, suppression of plantelongation, suppression of pollen growth, retention of freshness offlowers, use as an anti-stress agent for plants, weeds control,suppression of plant retrogradation, hypertrophism of roots and thelike.

The present invention is also directed to a method for regulating plantgrowth by inhibiting brassinosteroid metabolism by applying to a plant ametabolism inhibitor which comprises a compound represented by thefollowing formula (I) or a salt thereof

wherein R¹ and R² independently represent hydrogen atom or a lower alkylgroup, R³ represents hydrogen atom, a lower alkyl group or a loweralkoxyalkyl group, R⁴ represents a phenyl group which may besubstituted, X represents a single bond or —CH2—.

The present invention is also directed to a method for inhibitingbrassinosteroid metabolism by applying to a plant a metabolism inhibitorwhich comprises a compound represented by the following formula (I) or asalt thereof

wherein R¹ and R² independently represent hydrogen atom or a lower alkylgroup, R³ represents hydrogen atom, a lower alkyl group or a loweralkoxyalkyl group, R⁴ represents a phenyl group which may besubstituted, X represents a single bond or —CH2—.

R¹ and R² can be hydrogen atoms, R³ can be methyl group, R⁴ can4-(4-chlorophenyl)oxy-2-chlorophenyl group, and X can be a single bond.

R¹ and R² can be hydrogen atoms, R³ can be methyl group, R⁴ can bebiphenyl-4-yl group, and X can be a single bond.

R¹ and R² can be hydrogen atoms, R³ can be n-propyl group ormethoxymethyl group, R⁴ can be chlorophenyl group or methoxyphenylgroup, and X can be a single bond.

The brassinosteroid can be brassinolide.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the result of elongation of cress hypocotyl by themetabolism inhibitor of the present invention on day 5 after seeding.

FIG. 2 shows the result of elongation of cress hypocotyl by themetabolism inhibitor of the present invention on day 14 after seeding.

BEST MODE FOR CARRYING OUT THE INVENTION

The entire disclosures of the specification of Japanese PatentApplication No. 2000-225486 (filed on Jul. 26, 2000) are incorporated inthe disclosures of the present specification by reference.

In the aforementioned formula (I), as the lower alkyl group representedby R¹, R² or R³, a linear or branched alkyl group having 1 to about 6carbon atoms can be used (the same shall apply to a lower alkyl moietyof an alkoxy group or the like having the alkyl moiety). Examplesthereof include methyl group, ethyl group, n-propyl group, isopropylgroup, n-butyl group, sec-butyl group, tert-butyl group and the like. Itis preferred that both R¹ and R² are hydrogen atoms. Further, it is alsopreferred that R¹ and R² are hydrogen atoms, and R³ is a linear loweralkyl group (for example, methyl group, ethyl group, n-propyl group,n-butyl group and the like). It is particularly preferred that R¹ and R²are hydrogen atoms, and R³ is methyl group. Examples of the loweralkoxyalkyl group represented by R³ include, for example, methoxymethylgroup and the like. X is preferably a single bond.

When the phenyl group represented by R⁴ is substituted, types, numbersand substituting positions of substituents are not particularly limited.For example, the phenyl group may have preferably 1 to 3, morepreferably 1 or 2 of substituents. Where the phenyl group has two ormore substituents, they may be the same or different.

Examples of the substituent on the phenyl group include, for example, ahalogen atom (any of fluorine atom, chlorine atom, bromine atom andiodine atom), a lower alkyl group (methyl group, ethyl group and thelike), a lower cycloalkyl group (cyclopropyl group and the like), ahalogenated lower alkyl group (trifluoromethyl group and the like), alower alkoxy group (methoxy group, ethoxy group and the like), aminogroup, a mono- or dialkylamino group, carboxyl group, an alkoxycarbonylgroup (ethoxycarbonyl group and the like), an alkanoyl group (acetylgroup and the like), an aroyl group (benzoyl group and the like), anaralkyl group (benzyl group and the like), an aryl group (phenyl groupand the like), an aryloxy group (phenoxy group and the like), aheteroaryl group (pyridyl group and the like), a heteroaryloxy group(pyridyloxy group and the like), a heterocyclic group (pyrrolidinylgroup and the like), hydroxyl group, nitro group, cyano group and thelike. However, the substituents are not limited to these examples.

As the substituent on the phenyl group, a lower alkyl group, a halogenatom, a halogenated lower alkyl group, a lower alkoxy group, ahalogenated lower alkoxy group, hydroxyl group, an aryl group, and anaryloxy group are preferred. A halogen atom, a phenyl group (it mayfurther have one or more substituents on the ring), a phenoxy group (itmay further have one or more substituents on the ring) and a halogenatedlower alkyl group are more preferred. More preferred as the substituenton the phenyl group represented by R⁴ are one or more substituentsselected from the group consisting of a halogen atom, a substituted orunsubstituted phenyl group, and a substituted or unsubstituted phenoxygroup, and particularly preferred is a substituent selected from thegroup consisting of chlorine atom, phenyl group and 4-chlorophenoxygroup.

More specifically, examples of the substituted phenyl group representedby R⁴ include 2-chlorophenyl group, 4-chlorophenyl group,3,4-dichlorophenyl group, 2,4-dichlorophenyl group, 3,4-difluorophenylgroup, 2,4-difluorophenyl group, 4-bromophenyl group,4-trifluoromethoxyphenyl group, 4-toluyl group, 4-trifluoromethylphenylgroup, 3-trifluoromethylphenyl group, 4-hydroxyphenyl group,4-methoxyphenyl group, 2-chloro-4-trifluoromethylphenyl group,3-chloro-4-trifluoromethylphenyl group, 4-bromo-2-chlorophenyl group,biphenyl-4-yl group, (4-chlorophenyl)oxy-2-chlorophenyl group and thelike. Among them, biphenyl-4-yl group and4-(4-chlorophenyl)oxy-2-chlorophenyl group are preferred.

In the aforementioned formula (II), R¹¹ represents a lower alkyl group,a lower alkenyl group or a phenyl group which may be substituted. As thelower alkyl group, a linear or branched alkyl group having 1 to about 6carbon atoms can be used. Examples include methyl group, ethyl group,n-propyl group, isopropyl group, n-butyl group, sec-butyl group,tert-butyl group, isobutyl group and the like. Among them, methyl groupand ethyl group are preferred, and methyl group is particularlypreferred. As the lower alkenyl group, a linear or branched alkenylgroup having 2 to about 6 carbon atoms can be used. Examples includevinyl group, allyl group, 2-butenyl group and the like.

When the phenyl group represented by R¹¹ is substituted, types, numbersand substituting positions of substituents are not particularly limited.For example, the phenyl group may have preferably 1 to 3, morepreferably 1 or 2 of substituents. Where the phenyl group has 2 or moresubstituents, they may be the same or different. As the substituent onthe phenyl group, for example, any of those mentioned above can be used.

As the lower alkyl group or the phenyl group which may be substitutedrepresented by R¹² and the phenyl group which may be substitutedrepresented by R¹³, groups similar to each of those mentioned for thegroups represented by R¹¹ can be used. R¹² is preferably anunsubstituted phenyl group, and 2,4-difluorophenyl group and the likemay be used as a substituted phenyl group. Examples of the substitutedphenyl group represented by R¹³ include 4-chlorophenyl group and thelike.

The compounds represented by the aforementioned formula (I) or (II) mayhave one or more asymmetric carbon atoms. Optically active compounds anddiastereoisomers in pure forms based on the asymmetric carbon atoms aswell as any mixtures of the isomers (for example, mixtures of two ormore kinds of diastereoisomers), racemates and the like can be used asan active ingredient of the metabolism inhibitor of the presentinvention. Furthermore, the compounds represented by the aforementionedformula (I) or (II) can form acid addition salts, and may further formacid addition salts depending on the type of the substituent. The typesof the salts are not particularly limited, and examples of the saltsinclude salts with mineral acids such as hydrochloric acid, and sulfuricacid, salts with organic acids such as p-toluenesulfonic acid,methanesulfonic acid, and tartaric acid, metal salts such as sodiumsalts, potassium salts, and calcium salts, ammonium salts, salts withorganic amines such as triethylamine, salts with amino acids such asglycine, and the like.

Specific examples of the compounds represented by the formula (I)include those described in the specification of Japanese PatentApplication No. 2000-057564 as well as difenoconazole shown below,Brz401 and the like. Specific examples of the compounds represented bythe formula (II) include the following brassinazole as well as thecompounds described in the specifications of Japanese Patent UnexaminedPublication No. 2000-53657 and Japanese Patent Application No.2000-57565.

The compounds represented by the formula (I) can be prepared by themethods described in literature (for example, Zeitschrift furNaturforschung, 44c, pp. 85–96, 1989 and the like), or they areavailable as commercial products. For example, a compound represented bythe formula (I) wherein R¹ and R² are hydrogen atoms, R³ is n-propylgroup, R⁴ is 2,4-dichlorophenyl group, and X is a single bond isavailable from Ciba-Geigy as a fungicide (propiconazole). Furthermore,novel compounds can be prepared according to the methods described inliterature. The compounds represented by the formula (II) can be easilyprepared according to the methods described in Japanese PatentUnexamined Publication No. 2000-53657 and the specification of JapanesePatent Application No. 2000-57565.

The compounds represented by the formula (I) or (II) or salts thereof,as an active ingredient of the metabolism inhibitor of the presentinvention, have inhibitory action against the metabolism ofbrassinosteroids which are plant hormones. When the metabolism inhibitorof the present invention is applied to a plant, the inhibitor can exertthe same effect as that obtained when a brassinosteroid is administeredto a plant, and plant growth regulation based on the inhibition againstthe brassinosteroid metabolism can be achieved. The term “plant growthregulation” used in the specification should be construed in itsbroadest sense including, for example, regulation of plant elongationsuch as dwarfing of plants, pollen growth regulation, retention offlower freshness, use as a plant anti-stress agent (against heat,dryness, coldness or the like), weed control by regulation ofreproduction, suppression of plant retrogradation, control ofhypertrophy of root and the like. Furthermore, brassinosteroidsencompass compounds such as brassinolide, and the metabolism inhibitorof the present invention can inhibit the metabolism of any compoundencompassed in brassinosteroids.

The metabolism inhibitor of the present invention can be formulated, forexample, as an agricultural composition by using formulation additiveswell known in the art. Forms of the agricultural composition are notparticularly limited, and any forms that can be used in the art may bechosen. For example, compositions in the forms of emulsions, liquids,oils, water soluble powders, wettable powders, flowables, powders,subtilized granules, granules, aerosols, fumigants, pastes and the likecan be used. The methods for manufacturing the agricultural compositionare also not particularly limited, and any methods available to thoseskilled in the art can be appropriately employed. As the activeingredient of the metabolism inhibitor of the present invention, two ormore of the compounds represented by the aforementioned formula (I) or(II), or salts thereof, may be used in combination. Further, otheractive ingredients of agricultural chemicals such as insecticides,fungicides, insecticidal and fungicidal agents, herbicides and the likemay be incorporated.

Methods of application and doses of the metabolism inhibitor of thepresent invention can be suitably chosen by those skilled in the artdepending on conditions including a purpose of application, a dosageform, a plot to be treated and the like. The metabolism inhibitor of thepresent invention may sometimes inhibit brassinosteroid biosynthesiswhen used at a high concentration, and may exhibit an action on plantsopposite to the action of the metabolism inhibitor. Such a phenomenon isrecognized in difenoconazole, Brz217, Brz218, Brz224, Brz225 and thelike, which are typical examples of the metabolism inhibitor of thepresent invention. Therefore, the metabolism inhibitor of the presentinvention is preferably applied to plants at a relatively lowconcentration. However, concentrations to achieve optimum action can beappropriately determined by those skilled in the art by referring to thefollowing examples.

EXAMPLES

The present invention will be explained more specifically with referenceto examples. However, the scope of the present invention is not limitedto the following examples.

Example 1

Experimental Method For Growing Plants (Using Agar Medium)

This method was a common method for measurement of activity of each ofthe compounds. The activity as a metabolism inhibitor was measured as anactivity for accelerating elongation of cress hypocotyl. Seeds weresterilized by immersion in 1% hypochlorous acid for 20 minutes andwashed 5 times with sterilized water. Then, the seeds were sown on a 1%agar medium containing 0.5×MS medium and 1.5% sucrose, and grown inAgripot (purchased from Kirin Brewery Co. Ltd.) under sterilizedconditions. A test compound was prepared beforehand so as to be apredetermined concentration on this agar medium, and a control plot notadded with the compound was prepared for each of the tests. The plantswere grown at 25° C. under a cycle of 16-hour light and 8-hour darkconditions. After a predetermined growth period, activity determinationwas conducted by measuring the length of the cress lower hypocotyl.

As shown in FIGS. 1 and 2, when each compound was applied, accelerationof elongation of the cress hypocotyl was observed compared with thecontrol (in the figures, Dif represents difenoconazole, and thecompounds such as Brz217 are as described above). Further, whenbrassinolide was applied in combination, an acceleration tendency of theelongation was observed.

Example 2

To examine whether the accelerating activity on elongation of the cresshypocotyl was on the basis of the inhibitory activity against themetabolism of brassinolide, which is considered to be a practicallyactive brassinosteroid, activity of the compounds was examined indegradation system associated with oxidation of the 26th position ofbrassinolide. Immature seeds of Pisum sativum were used as plantmaterials. Brassinolide was added to a cell free system, and metabolicactivity was determined by quantifying the compound at the right end inthe following scheme to examine inhibitory action of difenoconazoleagainst metabolism.

TABLE 1 Ratio of metabolism inhibition Compound Concentration degree (%)relative to control Control 0 Difenoconazole 0.01 μM  0 Difenoconazole0.1 μM 7 Difenoconazole 1.0 μM 72  Difenoconazole  10 μM 76 

It was revealed that difenoconazole inhibited the metabolic process ofbrassinolide by 72% at a concentration of 1 μM. Although the results aredifferent from the test results in Example 1 in terms of effectiveconcentration, the difference is considered to be attributable to thedifference of plant species.

INDUSTRIAL APPLICABILITY

The compounds represented by the formula (I) or (II) or salts thereof,which are active ingredients of the metabolism inhibitors of the presentinvention, have inhibitory action against the brassinosteroidmetabolism, and can be used as plant growth regulators, for example, forsuppression of plant elongation, suppression of pollen growth, retentionof freshness of flowers, use as anti-stress agents for plants, weedscontrol, suppression of plant retrogradation, hypertrophism of roots andthe like.

1. A method for regulating plant growth by inhibiting brassinosteroidmetabolism by applying to a plant a metabolism inhibitor in aconcentration effective for plant growth regulation which comprises acompound represented by the following formula (I) or a salt thereof

wherein R¹ and R² independently represent hydrogen atom or a lower alkylgroup, R³ represents hydrogen atom, a lower alkyl group or a loweralkoxyalkyl group, R⁴ represents a phenyl group which may besubstituted, X represents a single bond or —CH2—.
 2. The methodaccording to claim 1, wherein R¹ and R² are hydrogen atoms, R³ is methylgroup, R⁴ is 4-(4-chlorophenyl)oxy-2-chlorophenyl group, and X is asingle bond.
 3. The method according to claim 1, wherein R¹ and R² arehydrogen atoms, R³ is methyl group, R⁴ is biphenyl-4-yl group, and X isa single bond.
 4. The method according to claim 1, wherein R¹ and R² arehydrogen atoms, R³ is n-propyl group or methoxymethyl group, R⁴ ischlorophenyl group or methoxyphenyl group, and X is a single bond. 5.The method according to claim 1, wherein the brassinosteroid isbrassinolide.
 6. The method according to claim 2, wherein thebrassinosteroid is brassinolide.
 7. The method according to claim 3,wherein the brassinosteroid is brassinolide.
 8. The method according toclaim 4, wherein the brassinosteroid is brassinolide.
 9. A method forinhibiting brassinosteroid metabolism by applying to a plant ametabolism inhibitor in a concentration effective for plant growthregulation which comprises a compound represented by the followingformula (I) or a salt thereof.

wherein R¹ and R² independently represent hydrogen atom or a lower alkylgroup, R³ represents hydrogen atom, a lower alkyl group or a loweralkoxyalkyl group, R⁴ represents a phenyl group which may besubstituted, X represents a single bond or —CH2—.
 10. The methodaccording to claim 9, wherein R¹ and R² are hydrogen atoms, R³ is methylgroup, R⁴ is 4-(4-chlorophenyl)oxy-2-chlorophenyl group, and X is asingle bond.
 11. The method according to claim 9, wherein R¹ and R² arehydrogen atoms, R³ is methyl group, R⁴ is biphenyl-4-yl group, and X isa single bond.
 12. The method according to claim 9, wherein R¹ and R²are hydrogen atoms, R³is n-propyl group or methoxymethyl group, R⁴ ischlorophenyl group or methoxyphenyl group, and X is a single bond. 13.The method according to claim 9, wherein the brassinosteroid isbrassinolide.
 14. The method according to claim 10, wherein thebrassinosteroid is brassinolide.
 15. The method according to claim 11,wherein the brassinosteroid is brassinolide.
 16. The method according toclaim 12, wherein the brassinosteroid is brassinolide.